Early responses in the Arabidopsis-Verticillium longisporum pathosystem are dependent on NDR1, JA- and ET-associated signals via cytosolic NPR1 and RFO1

The responses of Arabidopsis accessions and characterized genotypes were used to explore components in the early defense responses to the soilborne fungus Verticillium longisporum. V. longisporum susceptibility was found to be a complex trait, in which different disease phenotypes, such as stunting, altered flowering time, weight loss, and chlorosis were perceived differently across genotypes. A Bay-0 x Shahdara recombinant inbred line population was used to identify two loci on chromosomes 2 and 3 of Bay-0 origin that caused enhanced chlorosis after V. longisporum challenge. Furthermore, the observation that a mutation in RFO1 in Col-0 resulted in susceptibility whereas the natural rfo1 allele in Ty-0 showed a high degree of resistance to the pathogen supports the hypothesis that several resistance quantitative trait loci reside among Arabidopsis accessions. Analysis of mutants impaired in known pathogen response pathways revealed an enhanced susceptibility in ein2-1, ein4-1, ein6-1, esa1-1, and pad1-1, but not in other jasmonic acid (JA)-, ethylene (ET)-, or camalexin-deficient mutants, suggesting that V. longisporum resistance is regulated via a hitherto unknown JA- and ET-associated pathway. Pretreatments with the ET precursor 1-aminocyclo-propane-1-carboxylic acid (ACC) or methyl jasmonate (MeJA) caused enhanced resistance to V. longisporum. Mutants in the salicylic acid (SA) pathway (eds1-1, NahG, npr1-3, pad4-1, and sid2-1) did not show enhanced susceptibility to V. longisporum. In contrast, the more severe npr1-1 allele displayed enhanced V. longisporum susceptibility and decreased responses to ACC or MeJA pretreatments. This shows that cytosolic NPR1, in addition to SA responses, is required for JA- and ET-mediated V. longisporum resistance. Expression of the SA-dependent PR-1 and PR-2 and the ET-dependent PR-4 were increased 7 days postinoculation with V. longisporum. This indicates increased levels of SA and ET in response to V. longisporum inoculation. The R-gene signaling mutant ndr1-1 was found to be susceptible to V. longisporum, which could be complemented by ACC or MeJA pretreatments, in contrast to the rfo1 T-DNA mutant, which remained susceptible, suggesting that RFO1 (Fusarium oxysporum resistance) and NDR1 (nonrace specific disease resistance 1) activate two distinct signaling pathways for V. longisporum resistance.     

Identification of a disease resistance locus in Arabidopsis that is functionally homologous to the RPG1 locus of soybean

A new disease resistance locus in Arabidopsis, RPS3 , was identified using a previously cloned avirulence gene from a non- Arabidopsis pathogen. The avrB avirulence gene from the soybean pathogen Pseudomonas syringae pv. glycinea was transferred into a P. syringae pv. tomato strain that is virulent on Arabidopsis , and conversion to avirulence was assayed on Arabidopsis plants. The avrB gene had avirulence activity on most, but not all, Arabidopsis ecotypes. Of 53 ecotypes examined, 45 were resistant to a P. syringae pv. tomato strain carrying avrB , and eight were susceptible. The inheritance of this resistance was examined using crosses between the resistant ecotype Col-0 and the susceptible ecotype Bla-2. In F₂ plants from this cross, the ratio of resistant:susceptible plants was approximately 3:1, indicating that resistance to P. syringae expressing avrB is determined by a single dominant locus in ecotype Col-0, which we have designated RPS3 . Using RFLP analysis, RPS3 was mapped to chromosome 3, adjacent to markers M583 and G4523, and ≤ 1 cM from another disease resistance locus, RPM1 . In soybean, resistance to P. syringae strains that carry avrB is controlled by the locus RPG1 . Thus, RPG1 and RPS3 both confer avrB -specific disease resistance, suggesting that these genes may be homologs.     

The leucine-rich repeat domain can determine effective interaction between RPS2 and other host factors in arabidopsis RPS2-mediated disease resistance

Like many other plant disease resistance genes, Arabidopsis thaliana RPS2 encodes a product with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains. This study explored the hypothesized interaction of RPS2 with other host factors that may be required for perception of Pseudomonas syringae pathogens that express avrRpt2 and/or for the subsequent induction of plant defense responses. Crosses between Arabidopsis ecotypes Col-0 (resistant) and Po-1 (susceptible) revealed segregation of more than one gene that controls resistance to P. syringae that express avrRpt2. Many F(2) and F(3) progeny exhibited intermediate resistance phenotypes. In addition to RPS2, at least one additional genetic interval associated with this defense response was identified and mapped using quantitative genetic methods. Further genetic and molecular genetic complementation experiments with cloned RPS2 alleles revealed that the Po-1 allele of RPS2 can function in a Col-0 genetic background, but not in a Po-1 background. The other resistance-determining genes of Po-1 can function, however, as they successfully conferred resistance in combination with the Col-0 allele of RPS2. Domain-swap experiments revealed that in RPS2, a polymorphism at six amino acids in the LRR region is responsible for this allele-specific ability to function with other host factors.     

Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean.

To develop a model system for molecular genetic analysis of plant-pathogen interactions, we studied the interaction between Arabidopsis thaliana and the bacterial pathogen Pseudomonas syringae pv tomato (Pst). Pst strains were found to be virulent or avirulent on specific Arabidopsis ecotypes, and single ecotypes were resistant to some Pst strains and susceptible to others. In many plant-pathogen interactions, disease resistance is controlled by the simultaneous presence of single plant resistance genes and single pathogen avirulence genes. Therefore, we tested whether avirulence genes in Pst controlled induction of resistance in Arabidopsis. Cosmids that determine avirulence were isolated from Pst genomic libraries, and the Pst avirulence locus avrRpt2 was defined. This allowed us to construct pathogens that differed only by the presence or absence of a single putative avirulence gene. We found that Arabidopsis ecotype Col-0 was susceptible to Pst strain DC3000 but resistant to the same strain carrying avrRpt2, suggesting that a single locus in Col-0 determines resistance. As a first step toward genetically mapping the postulated resistance locus, an ecotype susceptible to infection by DC3000 carrying avrRpt2 was identified. The avrRpt2 locus from Pst was also moved into virulent strains of the soybean pathogen P. syringae pv glycinea to test whether this locus could determine avirulence on soybean. The resulting strains induced a resistant response in a cultivar-specific manner, suggesting that similar resistance mechanisms may function in Arabidopsis and soybean.     

Transgressive segregation reveals two Arabidopsis TIR-NB-LRR resistance genes effective against Leptosphaeria maculans, causal agent of blackleg disease

In a cross between the two resistant accessions Col-0 and Ler-0, a 15:1 segregation was found in F2, suggesting the presence of unlinked resistance loci to Leptosphaeria maculans. One hundred Col-4 x Ler-0, and 50 Ler-2 x Cvi-1 recombinant inbred lines, and seven susceptible Ler-0 x Ws-0 F2 progenies were examined to identify the two loci. Resistance in Col-4, Ws-0 and Cvi-1 (RLM1) was mapped to the marker m305 on chromosome 1. Col-4 x Ler-0 and Ler-2 x Cvi-1 mapping populations located RLM2(Ler) on the same arm of chromosome 4. A tight physical location of RLM2 was established through near-isogenic lines. This region was found to correspond to an ancient duplication event between the RLM1 and RLM2 loci. Two independent T-DNA mutants in a TIR-NB-LRR R gene (At1g64070) displayed susceptibility, and L. maculans susceptible mutant phenotypes were confirmed to be allelic for rlm1 in F1 after crosses with susceptible rlm1(Ler)rlm2(Col) plants. Complementation of rlm1(Ler)rlm2(Col) with the genomic Col-0 sequence of At1g64070 conferred resistance. In addition, two T-DNA mutants in a neighbouring homologous TIR-NB-LRR gene (At1g63880) displayed moderate susceptibility to L. maculans. Sequence analysis revealed that At1g64070 was truncated by a premature stop codon, and that At1g63880 was absent in Ler-0. RNA interference confirmed that Ler-0 resistance is dependent on genes structurally related to RLM1. Camalexin was identified as a quantitative co-dominant resistance factor of Col-0 origin, but independent of RLM1. RLM1/RLM2 resistance was, however, found to require RAR1 and partially HSP90.1.     

The mitochondrial genome of Fusarium oxysporum

Physical and genetic maps have been constructed for mtDNA from strains of the fungus Fusarium oxysporum representing three pathogenically specialized forms. All three mtDNA maps are circular. Their sizes are 45 kb for F. oxysporum f.sp. raphani and 52 kb for both F. oxysporum f.sp. conglutinans and F. oxysporum f.sp. matthioli. The genetic loci for cytochrome b, the mitochondrial 25S ribosomal RNA and cytochrome oxidase subunit II, have been identified and are similarly arranged on the three genomes.     

RCH1, a locus in Arabidopsis that confers resistance to the hemibiotrophic fungal pathogen Colletotrichum higginsianum

When challenged with the crucifer pathogen Colletotrichum higginsianum, Arabidopsis thaliana ecotype Columbia (Col-0) was colonized by the fungus within 2 to 3 days, developing brown necrotic lesions surrounded by a yellow halo. Lesions spread from the inoculation site within 3 to 4 days, and subsequently continued to expand until they covered the entire leaf. Electron microscopy confirmed that C. higginsianum is a hemibiotroph on Arabidopsis, feeding initially on living cells as a biotroph before switching to a necrotrophic mode of growth. A collection of 37 ecotypes of Arabidopsis varied in their responses to infection by C. higginsianum. The ecotype Eil-0 was highly resistant, with symptoms limited to necrotic flecking and with only very limited fungal colonization. Analyses suggested that the hypersensitive response and reactive oxygen species may be important in this defense response. Expression analyses with cDNA microarrays indicated that the defense reaction depends primarily on the jasmonic acid- and ethylene-dependent signaling pathways and, to a lesser extent, on the salicylate-dependent pathway. Crosses between the Eil-0 and Col-0 ecotypes suggested that the resistance in Eil-0 was dominant and was conferred by a single locus, which we named RCH1. RCH1 is the first resistance locus to be identified from Arabidopsis against the hemibiotrophic fungus genus Colletotrichum.     

The Arabidopsis thaliana DNA-binding protein AHL19 mediates verticillium wilt resistance

Verticillium spp. are destructive soilborne fungal pathogens that cause vascular wilt diseases in a wide range of plant species. Verticillium wilts are particularly notorious, and genetic resistance in crop plants is the most favorable means of disease control. In a gain-of-function screen using an activation-tagged Arabidopsis mutant collection, we identified four mutants, A1 to A4, which displayed enhanced resistance toward the vascular wilt species Verticillium dahliae, V. albo-atrum and V. longisporum but not to Fusarium oxysporum f. sp. raphani. Further testing revealed that mutant A2 displayed enhanced Ralstonia solanacearum resistance, while mutants A1 and A3 were more susceptible toward Pseudomonas syringae pv. tomato. Identification of the activation tag insertion site in the A1 mutant revealed an insertion in close proximity to the gene encoding AHL19, which was constitutively expressed in the mutant. AHL19 knock-out alleles were found to display enhanced Verticillium susceptibility whereas overexpression of AHL19 resulted in enhanced Verticillium resistance, showing that AHL19 acts as a positive regulator of plant defense.     

Evolutionary trade-offs at the Arabidopsis WRR4A resistance locus underpin alternate Albugo candida race recognition specificities

The oomycete Albugo candida causes white rust of Brassicaceae, including vegetable and oilseed crops, and wild relatives such as Arabidopsis thaliana. Novel White Rust Resistance (WRR) genes from Arabidopsis enable new insights into plant/parasite co-evolution. WRR4A from Arabidopsis accession Columbia (Col-0) provides resistance to many but not all white rust races, and encodes a nucleotide-binding, leucine-rich repeat immune receptor. Col-0 WRR4A resistance is broken by AcEx1, an isolate of A. candida. We identified an allele of WRR4A in Arabidopsis accession Øystese-0 (Oy-0) and other accessions that confers full resistance to AcEx1. WRR4A^Oy-0 carries a C-terminal extension required for recognition of AcEx1, but reduces recognition of several effectors recognized by the WRR4A^Col-0 allele. WRR4A^Oy-0 confers full resistance to AcEx1 when expressed in the oilseed crop Camelina sativa.     

Genetic and physiological analysis of the relationship between partial resistance to clubroot and tolerance to trehalose in Arabidopsis thaliana

In Arabidopsis thaliana the induction of plant trehalase during clubroot disease was proposed to act as a defense mechanism in the susceptible accession Col-0, which could thereby cope with the accumulation of pathogen-synthesized trehalose. In the present study, we assessed trehalose activity and tolerance to trehalose in the clubroot partially resistant accession Bur-0. We compared both accessions for several trehalose-related physiological traits during clubroot infection. A quantitative trait loci (QTLs) analysis of tolerance to exogenous trehalose was also conducted on a Bur-0xCol-0 RIL progeny. Trehalase activity was not induced by clubroot in Bur-0 and the inhibition of trehalase by validamycin treatments resulted in the enhancement of clubroot symptoms only in Col-0. In pathogen-free cultures, Bur-0 showed less trehalose-induced toxicity symptoms than Col-0. A QTL analysis identified one locus involved in tolerance to trehalose overlapping the confidence interval of a QTL for resistance to Plasmodiophora brassicae. This colocalization was confirmed using heterogeneous inbred family (HIF) lines. Although not based on trehalose catabolism capacity, partial resistance to clubroot is to some extent related to the tolerance to trehalose accumulation in Bur-0. These findings support an original model where contrasting primary metabolism-related regulations could contribute to the partial resistance to a plant pathogen.     

Genetic analysis of developmentally regulated resistance to downy mildew (Hyaloperonospora parasitica) in Arabidopsis thaliana

Although developmentally regulated disease resistance has been observed in a variety of plant-pathogen interactions, the molecular basis of this phenomenon is not well understood. Arabidopsis thaliana ecotype Columbia-0 (Col-0) expresses a developmentally regulated resistance to Hyaloperonospora parasitica isolate Emco5. Col-0 seedlings support profuse mycelial growth and asexual spore formation in the cotyledons. In contrast, Emco5 growth and reproduction is dramatically (but not completely) restricted in the first set of true leaves. Subsequent leaves exhibit progresssively increased resistance. This adult resistance is strongly suppressed by expression of the salicylic acid-degrading transgene NahG and by loss-of-function mutations in the defense-response regulators PAD4, NDR1, RAR1, PBS3, and NPR1. In contrast to Col-0, the Wassilewskija-0 (Ws-0) ecotype supports profuse growth of Emco5 at all stages of development. Gene-dosage experiments and segregation patterns indicate that adult susceptibility in Ws-0 is incomepletely dominant to adult resistance in Col-0. Genetic mapping in a Col x Ws F2 population revealed a major locus on the bottom arm of chromosome 5, which we named RPP31. Analysis of T-DNA insertion lines indicated that the Columbia allele of RPP8, though tightly linked to RPP31, is not necessary for adult resistance.     

ERECTA, an LRR receptor-like kinase protein controlling development pleiotropically affects resistance to bacterial wilt

Bacterial wilt, one of the most devastating bacterial diseases of plants worldwide, is caused by Ralstonia solanacearum and affects many important crop species. We show that several strains isolated from solanaceous crops in Europe are pathogenic in different accessions of Arabidopsis thaliana. One of these strains, 14.25, causes wilting symptoms in A. thaliana accession Landsberg erecta (Ler) and no apparent symptoms in accession Columbia (Col-0). Disease development and bacterial multiplication in the susceptible Ler accession depend on functional hypersensitive response and pathogenicity (hrp) genes, key elements for bacterial pathogenicity. Genetic analysis using Ler x Col-0 recombinant inbred lines showed that resistance is governed by at least three loci: QRS1 (Quantitative Resistance to R. solanacearum) and QRS2 on chromosome 2, and QRS3 on chromosome 5. These loci explain about 90% of the resistance carried by the Col-0 accession. The ERECTA gene, which encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) and affects development of aerial organs, is dimorphic in our population and lies close to QRS1. Susceptible Ler plants transformed with a wild-type ERECTA gene, and the LER line showed increased disease resistance to R. solanacearum as indicated by reduced wilt symptoms and impaired bacterial growth, suggesting unexpected cross-talk between resistance and developmental pathways.     

Identification and characterization of victorin sensitivity in Arabidopsis thaliana

Cochliobolus victoriae is a necrotrophic fungus that produces a host-selective toxin called victorin. Victorin is considered to be host selective because it has been known to affect only certain allohexaploid oat cultivars containing the dominant Vb gene. Oat cultivars containing Vb are also the only genotypes susceptible to C. victoriae. Assays were developed to screen the "nonhost" plant of C. victoriae, Arabidopsis thaliana, for victorin sensitivity. Sensitivity to victorin was identified in six of 433 bulk populations of Arabidopsis. In crosses of Col-4 (victorin-insensitive) x victorin-sensitive Arabidopsis ecotypes, victorin sensitivity segregated as a single dominant locus, as it does in oats. This Arabidopsis locus was designated LOV, for locus orchestrating victorin effects. Allelism tests indicate that LOV loci are allelic or closely linked in all six victorin-sensitive ecotypes identified. LOV was localized to the north arm of Arabidopsis thaliana chromosome I. The victorin-sensitive Arabidopsis line LOV1 but not the victorin-insensitive line Col-4 was susceptible to C. victoriae infection. Consequently, the LOV gene appears to be a genetically dominant, disease susceptibility gene.     

Overexpression of an auxilin-like gene (F9E10.5) can suppress Al uptake in roots of Arabidopsis

Plants resistant to aluminium (Al) stress were isolated from Arabidopsis thaliana enhancer-tagged mutant lines. Compared with the parental Col-7 control line, one of the resistant candidates, #355-2, showed a higher expression of the F9E10.5 gene (At1g75100) on chromosome 1, a lower Al content in whole roots, and a shorter root hair length (approximately 30%). Both Al influx and associated oxidative stress occurred in root hairs, as well as in root tips of Col-7; however, they were seen only in root tips of #355-2. Transgenic plants overexpressing the F9E10.5 gene showed a slightly higher Al resistance than their parental control line (Ler). The F9E10.5 gene encodes an auxilin-like protein related to the clathrin-uncoating process in endocytosis. Microscopic observation indicated that both Al ion influx and endocytosis activity were lower in root hair cells of the #355-2 line than in those of Col-7. These results suggested that overexpression of this auxilin-like protein inhibits endocytosis in root hair cells by a disturbance of the transport system as in animal cells shown previously. It was also suggested that a part of the Al influx occurred via endocytosis in root hair cells in Arabidopsis. The Al resistance in the #355-2 line may therefore be due to a lower Al uptake via endocytosis in the root hair region.     

Identification and characterization of the SSB1 locus involved in symptom development by Spring beauty latent virus infection in Arabidopsis thaliana

The natural variation of Arabidopsis thaliana in response to a bromovirus, Spring beauty latent virus (SBLV), was examined. Of 63 Arabidopsis accessions tested, all were susceptible when inoculated with SBLV, although there was a large degree of variation in symptom development. Most accessions, including Columbia (Col-0), were symptomless or developed only mild symptoms, but four accessions, including S96, showed severe symptoms of SBLV infection. Genetic analysis suggested that the difference in the responses of Col-0 and S96 to SBLV was controlled by a single semidominant locus. We have designated this locus SSB1 (symptom development by SBLV infection). By using genetic markers, SSB1 was mapped to chromosome IV. The patterns of distribution and accumulation of SBLV in sensitive accessions were similar to those in the insensitive accessions. In addition, symptom development in S96 by SBLV infection was critically interrupted by the presence of the NahG gene, which encodes salicylic acid (SA) hydroxylase. These data suggest that symptom development in A. thaliana controlled by SSB1 is independent of the efficiency of SBLV multiplication and is dependent on SA signaling.     

Arabidopsis is susceptible to the cereal ear blight fungal pathogens Fusarium graminearum and Fusarium culmorum

The fungal pathogens Fusarium graminearum and F. culmorum cause ear blight disease on cereal crops worldwide. The disease lowers both grain quality and grain safety. Disease prevalence is increasing due to changes in cropping practices and the difficulties encountered by plant breeders when trying to introgress the polygene-based resistance. The molecular basis of resistance to Fusarium ear blight in cereal species is poorly understood. This is primarily due to the large size of cereal genomes and the expensive resources required to undertake gene function studies in cereals. We therefore explored the possibility of developing various model floral infection systems that would be more amenable to experimental manipulation and high-throughput gene function studies. The floral tissues of tobacco, tomato, soybean and Arabidopsis were inoculated with Fusarium conidia and this resulted in disease symptoms on anthers, anther filaments and petals in each plant species. However, only in Arabidopsis did this initial infection then spread into the developing siliques and seeds. A survey of 236 Arabidopsis ecotypes failed to identify a single genotype that was extremely resistant or susceptible to Fusarium floral infections. Three Arabidopsis floral mutants that failed to develop anthers and/or functional pollen (i.e. agamous-1, apetala1-3 and dad1) were significantly less susceptible to Fusarium floral infection than wild type. Deoxynivalenol (DON) mycotoxin production was also detected in Fusarium-infected flowers at >1 ppm. This novel floral pathosystem for Arabidopsis appears to be highly representative of a serious cereal crop disease.     

Rapid activation of a novel plant defense gene is strictly dependent on the Arabidopsis RPM1 disease resistance locus.

We cloned and sequenced cDNAs encoded by a novel plant defense gene, ELI3, from parsley and Arabidopsis thaliana. The predicted product shares no homology to known sequences. ELI3 mRNA accumulates in A. thaliana leaves in response to challenge with phytopathogenic Pseudomonas syringae strains. The timing and magnitude of this response are dictated by the genetics of the plant-pathogen interaction being analyzed. During incompatible interactions, where resistance in the plant genotype Col-0 is dictated by the dominant RPM1 locus, ELI3 mRNA accumulates to high levels 5-10 h post-inoculation. This kinetic behavior is also generated by the presence of a cloned bacterial avirulence gene, in otherwise virulent bacteria, which triggers resistance mediated via RPM1 action. The phenotypic outcome is a hypersensitive resistance reaction visible 8-15 h post-infiltration. Thus, the induction kinetics of ELI3 mRNA accumulation are consistent with a functional role for the ELI3 gene product in establishing the resistant phenotype. In contrast, during compatible interactions with the susceptible plant genotype Nd-0, which is homozygous recessive at the rpm1 locus, ELI3 mRNA accumulates significantly only after 15 h. We show genetically that ELI3 activation is strictly dependent on the presence of dominant alleles at RPM1 using an assay generalizable to any pathogen induced plant defense phenomena.